Summary: 1. In collaboration with Dr. Robert Reynolds of the Southern Research Institute, we are trying to identify the intracellular targets for 2 very active novel antituberculosis agents. When the drug targets are identified, we will be involved in the design of even better antituberculosis compounds using molecular modeling and combitorial chemistry techniques. The general approach is to generate resistant clones, create gene libraries from chromosomal DNA from these clones, transform drug sensitive cells with these libraries, and then search for resistance genes by plating transformants on drug selection media. For the initial studies, we have chosen to evaluate drug resistance in M. smegmatis because this mycobacterial strain is much easier and safer to work with than M. tuberculosis. Thus far, we have isolated several M. smegmatis clones that are highly resistant to the two pyrido-pyrazine carbamic acid compounds. We have also isolated DNA from resistant organisms and have created plasmid libraries. We are in the process of transforming drug sensitive cells with the resistance libraries and selecting for drug resistant transformants. 2. In collaboration with Dr. Larry Bockstahler from FDA/CDRH, we have been attempting to develop rapid and sensitive molecular methods to detect drug-resistant M. tuberculosis organisms. These methods are based on the identification of specific mutations in genes known to be associated with drug resistance in M. tuberculosis. Our initial efforts involved using peptide nucleic acid (PNA) probes to detect gene mutations. PNA probes are useful for detecting mutant nucleic acids, because they have high thermal stability, strong binding capacity and high binding specificity. Using a PNA-PCR-ELISA format, we have shown that we can quickly and specifically identify mutations in the M. tuberculosis katG (isoniazid resistance) and rpoB genes (rifampin resistance)that are known to be associated with drug resistance. Recently, we have extended this approach for quickly identifying TB mutations to a microarray format. We have shown that certain mutations in the katG and rpoB genes can be identified using the microarray technology. We are in the process of establishing the conditions to identify other mutations in TB genes associated with drug resistance.